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Effects of intravenous ethyl pyruvate on cardiopulmonary variables and quality of recovery from anesthesia in horses

Published:February 07, 2022DOI:https://doi.org/10.1016/j.vaa.2022.01.008

      Abstract

      Objective

      To determine the effects of intravenous ethyl pyruvate, an anti-inflammatory with putative benefits in horses with endotoxemia, on cardiopulmonary variables during anesthesia and the quality of anesthetic recovery.

      Study design

      Randomized, crossover, blinded experimental design.

      Animals

      A total of six healthy Standardbred geldings, aged 13 ± 3 years and weighing 507 ± 66 kg (mean ± standard deviation).

      Methods

      Horses were anesthetized for approximately 90 minutes on two occasions with a minimum of 2 weeks apart using xylazine for sedation, ketamine and diazepam for induction, and isoflurane in oxygen for maintenance. Lactated Ringer’s solution (LRS; 10 mL kg–1 hour–1) was administered during anesthesia. Treatments were randomized and administered starting approximately 30 minutes after induction of anesthesia and infused over 60 minutes: LRS (1 L) or ethyl pyruvate (150 mg kg–1 in 1 L LRS). Invasive arterial pressures, heart rate, respiratory rate and end-tidal carbon dioxide tensions were recorded every 5 minutes for the duration of anesthesia. Arterial blood gases, glucose and lactate concentrations were measured every 20 minutes. Anesthetic recovery was video recorded, stored, and subsequently rated by two individuals blinded to treatments. Total recovery time, time to extubation, number of attempts and time to sternal recumbency, number of attempts to stand and time to stand were recorded. Quality of recovery was analyzed. Data between treatments and within a treatment were assessed using two-way repeated-measures anova and a Pearson correlation coefficient, significant at p < 0.05.

      Results

      All horses completed the study. No significant differences were detected between the ethyl pyruvate and LRS treatments for either the cardiopulmonary variables or quality of recovery from anesthesia.

      Conclusions and clinical relevance

      The results suggest that intravenous ethyl pyruvate can be administered to healthy anesthetized horses with minimal impact on the cardiopulmonary variables studied or the quality of recovery from anesthesia.

      Keywords

      Introduction

      Equine anesthesia is associated with a higher morbidity and mortality rate compared with small animals and humans.
      • Johnston G.M.
      • Eastment J.K.
      • Wood J.L.N.
      • Taylor P.M.
      The confidential enquiry into perioperative equine fatalities (CEPEF): mortality results of Phases 1 and 2.
      reported an overall mortality rate of 1.9% in horses. That study found that 33% of deaths were of cardiovascular origin and 32% followed fractures and myopathies. Almost 18 years later in another study, 92% of anesthetized horses that died did so during the recovery period associated with neuromuscular (46.9%), respiratory (22.6%), cardiovascular (13.6%) and systemic (15.8%) complications (
      • Laurenza C.
      • Ansart L.
      • Portier K.
      Risk factors of anesthesia-related mortality and morbidity in one equine hospital: a retrospective study on 1,161 cases undergoing elective or emergency surgeries.
      ). By contrast, mortality associated with anesthesia in sick dogs, cats and rabbits was 1.33%, 1.40% and 7.37%, respectively (
      • Brodbelt D.V.
      • Blissitt K.J.
      • Hammond R.A.
      • et al.
      The risk of death: the confidential enquiry into perioperative small animal fatalities.
      ), and in humans it was 0.01% (
      • Lunn J.N.
      • Mushin W.W.
      Mortality associated with anaesthesia.
      ).
      Horses anesthetized for abdominal exploratory surgery related to colic may have significant cardiopulmonary compromise from a variety of causes including abdominal distention, dehydration and hypovolemia, tissue ischemia and pain. Large colon volvulus and small intestine strangulation are two potentially devastating forms of colic resulting in high morbidity, mortality and client expense (
      • Mair T.S.
      • Edwards G.B.
      Strangulating obstructions of the small intestine.
      ;
      • Ellis C.M.
      • Lynch T.M.
      • Slone D.E.
      • et al.
      Survival and complications after large colon resection and end-to-end anastomosis for strangulating large colon volvulus in seventy-three horses.
      ). Frequently, oxygen delivery to tissues is critically reduced by the disease process and compounded by the effects of anesthetic drugs (
      • Cambier C.
      • Wierinckx M.
      • Grulke S.
      • et al.
      The effect of colic on oxygen extraction in horses.
      ). The development of an effective therapeutic strategy that diminishes ischemic injury and hastens repair could improve systemic perfusion and oxygenation during anesthesia and may positively impact survival.
      Previous studies have examined different anesthetic protocols to make the recovery period safer for the horses and personnel. These drugs, including α2-adrenergic agonists, propofol and ketamine, have been used to prolong the duration of recumbency during recovery, allowing increased elimination of inhalant agents and improved limb coordination and behavior when attempting to stand (
      • Santos M.
      • Fuente M.
      • Garcia-Iturralde P.
      • et al.
      Effects of α2-adrenoceptor agonists during recovery from isoflurane anaesthesia in horses.
      ;
      • Steffey E.P.
      • Mama K.R.
      • Brosnan R.J.
      • et al.
      Effect of administration of propofol and xylazine hydrochloride on recovery of horses after four hours of anesthesia with desflurane.
      ;
      • Wagner A.E.
      • Mama K.R.
      • Steffey E.P.
      • Hellyer P.W.
      Evaluation of infusions of xylazine with ketamine or propofol to modulate recovery following sevoflurane anesthesia in horses.
      ;
      • Woodhouse K.J.
      • Brosnan R.J.
      • Nguyen K.Q.
      • et al.
      Effects of postanesthetic sedation with romifidine or xylazine on quality of recovery from isoflurane anesthesia in horses.
      ).
      Ethyl pyruvate (EP) is a prometabolic antioxidant with diverse pharmacological effects (
      • Olcum M.
      • Tufekci K.U.
      • Durur D.Y.
      • et al.
      Ethyl pyruvate attenuates microglial NLRP3 inflammasome activation via inhibition of HMGB1/NF-κB/miR-223 signaling.
      ). EP diminished clinical signs of endotoxemia and expression of proinflammatory genes in horses when administered at a dosage of 150 mg kg–1, infused in 1 L of lactated Ringer’s solution (LRS) over 60 minutes (
      • Schroeder E.L.
      • Holcombe S.J.
      • Cook V.L.
      • et al.
      Preliminary safety and biological efficacy studies of ethyl pyruvate in normal mature horses.
      ;
      • Jacobs C.C.
      • Holcombe S.J.
      • Cook V.L.
      • et al.
      Ethyl pyruvate diminishes the inflammatory response to lipopolysaccharide infusion in horses.
      ). EP blocks nuclear factor-kappaB (NF-κB) DNA binding, thereby reducing proinflammatory cytokines in systemic circulation in mice and horses (
      • Ulloa L.
      • Ochani M.
      • Yang H.
      • et al.
      Ethyl pyruvate prevents lethality in mice with established lethal sepsis and systemic inflammation.
      ;
      • Schroeder E.L.
      • Holcombe S.J.
      • Cook V.L.
      • et al.
      Preliminary safety and biological efficacy studies of ethyl pyruvate in normal mature horses.
      ;
      • Albensi B.C.
      What is nuclear factor kappa B (NF-κB) doing in and to the mitochondrion?.
      ). EP has also been shown to reduce neutrophil infiltration in the airway of mice by downregulating high mobility group box-1, which has been identified as an inflammatory mediator (
      • Tang H.
      • Zhao H.
      • Song J.
      • et al.
      Ethyl pyruvate decreases airway neutrophil infiltration partly through a high mobility group box 1-dependent mechanism in a chemical-induced murine asthma model.
      ). A previous study found no negative impact of EP administered postoperatively to horses after surgical correction of a large colon volvulus (
      • Johnson L.M.
      • Holcombe S.J.
      • Shearer T.R.
      • et al.
      Multicenter placebo-controlled randomized study of ethyl pyruvate in horses following surgical treatment for ≥360° large colon volvulus.
      ). In dogs with septic shock, EP improved perfusion and oxygenation of tissues (
      • Kou Q.Y.
      • Guan X.D.
      [Effect of ethyl pyruvate on indices of tissue oxygenation and perfusion in dogs with septic shock].
      ). During hemorrhagic episodes in pigs, EP provided anti-inflammatory benefits where it modulated splenic NF-κB (
      • Dong W.
      • Cai B.
      • Peña G.
      • et al.
      Ethyl pyruvate prevents inflammatory responses and organ damage during resuscitation in porcine hemorrhage.
      ). Furthermore, EP was shown to act as an anti-inflammatory agent in rats with uveitis and as a reactive oxygen species scavenger in human patients with multiple organ injuries (
      • Fedeli D.
      • Falcioni G.
      • Olek R.A.
      • et al.
      Protective effect of ethyl pyruvate on msP rat leukocytes damaged by alcohol intake.
      ;
      • Yang R.
      • Zhu S.
      • Tonnessen T.I.
      Ethyl pyruvate is a novel anti-inflammatory agent to treat multiple inflammatory organ injuries.
      ).
      The adoption of a novel therapeutic, such as EP, should be avoided in anesthetized horses without prior knowledge about the effects on cardiopulmonary function and the quality of recovery from anesthesia. Horses with colic may have physiologic and metabolic abnormalities that alter responses to anesthetic drugs and the quality of recovery from anesthesia. Adverse effects may preclude use of a new therapeutic agent in the perianesthetic period.
      Drugs that improve cardiac output (CO), oxygen delivery and central venous pressure during anesthesia may hasten the elimination of anesthetic drugs and improve the ability of the horse to safely stand (
      • Taylor M.D.
      • Grand T.J.
      • Cohen J.E.
      • et al.
      Ethyl pyruvate enhances ATP levels, reduces oxidative stress and preserves cardiac function in a rat model of off-pump coronary bypass.
      ). However, because of its prometabolic property, there is a concern that use of EP may substantially increase the elimination of inhalant or injectable drugs, which could result in a horse attempting to stand early after anesthesia. This effect could negatively affect the quality of the recovery and increase the risk of injury. Therefore, the aim of the present study was to determine the effect of EP administration on cardiopulmonary variables and recovery in isoflurane anesthetized horses. Our hypothesis was that EP would not negatively affect the cardiopulmonary variables measured during general anesthesia or the quality of recovery in healthy horses.

      Materials and methods

      The study was approved by the Michigan State University (MSU) Institutional Animal Care and Use Committee (no. 08/17-134-00). A total of six retired adult Standardbred geldings from the MSU teaching herd were enrolled in the study. Horses were healthy and had no limb injuries or lameness based on a physical examination that was performed upon their arrival in the hospital 18–24 hours prior to the study. Horses were admitted as pairs to decrease stress during transport and hospitalization, and housed in 3 × 3.6 m box stalls. The horses were aged 13 ± 3 years and weighed 507 ± 66 kg, mean ± standard deviation (SD). Inclusion criteria included horses with no symptoms of systemic disease, no significant musculoskeletal lameness and an American Society of Anesthesiologists (ASA) classification of I. Horses that were difficult to handle or with a history of non-steroidal anti-inflammatory drug administration within 24 hours were excluded. Grain and hay were withheld for 12 and 4 hours, respectively, prior to the study, and water was available without restriction until premedication.
      Horses were assigned to one of two treatments using a random number generator (Microsoft Excel for Windows; Microsoft, WA, USA): lactated Ringers solution (treatment LRS) or ethyl pyruvate (treatment EP) for the first anesthesia episode, and the alternate treatment was administered on the second anesthesia episode conducted a minimum of 2 weeks later.

      Catheter placement and sedation

      On the day of the study, a physical examination was performed and recorded. The skin over an external jugular vein was aseptically prepared and 1 mL of 2% lidocaine (MWI Animal Health, ID, USA) was injected subcutaneously at the insertion site of the intravenous (IV) catheter. A 14 gauge, 13 cm IV catheter (MILA International Inc., KY, USA) was then placed in the jugular vein and sutured to the skin. Horses were walked to an anesthesia preparation area and administered xylazine (0.4 mg kg–1; Akorn Inc., IL, USA) IV.

      Induction and maintenance of anesthesia

      Horses were moved to an induction stall and administered additional xylazine (0.4 mg kg–1) IV. Anesthesia was induced using ketamine (2.2 mg kg–1; Akorn Inc.) IV and diazepam (0.08 mg kg–1; Akorn Inc.) IV when the horses appeared sedated. A squeeze-gate system was used to assist with induction. The horse was orally intubated in lateral recumbency by placing a cylindrical bite block between the incisors and inserting a 26 mm internal diameter endotracheal tube (Surgivet; Smiths Medical, OH, USA) into the trachea. The horses were lifted using a hoist and hobble system and placed on a padded surgery table in dorsal recumbency. Horses at a light plane of anesthesia (as evident by nystagmus or movement of their limbs or neck) were administered additional doses of IV ketamine (0.20–0.25 mg kg–1). The horses were connected to a circle delivery system (Tafonius; Hallowell Engineering & Manufacturing Corp., MA, USA). Isoflurane was initially administered at a vaporizer setting of 3% with 8 L minute–1 oxygen for 15–20 minutes. The vaporizer setting was then reduced to 2% and oxygen to 4 L minute–1 for the remainder of the study. Anesthetic depth was determined based on clinical signs, such as nystagmus, increased muscle tone, increased blood pressure and spontaneous movement (
      • Nannarone S.
      • Spadavecchia C.
      Evaluation of the clinical efficacy of two partial intravenous anesthetic protocols, compared with isoflurane alone, to maintain general anesthesia in horses.
      ). Controlled ventilation was maintained at a respiratory frequency (fR) of 8–9 breaths minute–1 and a tidal volume of 15 mL kg–1 minute–1. LRS (10 mL kg–1 hour–1) was continued IV during anesthesia. Dobutamine hydrochloride (1–5 μg kg–1 minute–1; Hospira Inc., IL, USA) was infused IV as needed to maintain mean arterial blood pressure (MAP) >70 mmHg. Hypoxemia was defined as a partial pressure of arterial oxygen (PaO2) <70 mmHg (9 kPa). Treatment was aerosolized salbutamol (2 μg kg–1; Albuterol sulfate; GlaxoSmithKline, UK) injected through an adapter on the Y-piece of the rebreathing circuit (
      • Robertson S.A.
      • Bailey J.E.
      Aerosolized salbutamol (albuterol) improves PaO2 in hypoxaemic anaesthetized horses — a prospective clinical trial in 81 horses.
      ). The number of times that salbutamol was administered was recorded.

      Infusion

      Starting approximately 30 minutes after the induction of anesthesia, an infusion of either 1 L of LRS (treatment LRS) or 150 mg kg–1 EP (E47808-1000G; Sigma-Aldrich, MO, USA) in 1 L of LRS (treatment EP) was administered over 60 minutes (Fig. 1). Infusions were administered using a Medfusion 3500 syringe pump (Smiths Medical ASD, MN, USA). Everyone involved in the study, including the anesthetist, was blinded to the assigned treatment except for the person (SJH) who administered the treatments. At the end of this infusion, isoflurane was discontinued and the horse was moved to a recovery stall measuring 2.9 × 3.4 m.
      Figure 1
      Figure 1Study timeline for six horses that were administered an infusion of lactated Ringer’s solution or ethyl pyruvate over 60 minutes, starting 30 minutes after induction of anesthesia. Cardiopulmonary variables were recorded during the infusions and the quality of recovery from anesthesia was scored. Gray arrows, heart rate and arterial pressures recorded; black arrows, arterial blood collected for measurements of glucose and lactate concentrations, pH and blood gases.

      Data collection, sampling and sample analysis

      After induction of general anesthesia, a 20 gauge, 2.5 cm catheter (BD Medical, UT, USA) was placed in the left facial artery. A blood pressure transducer (Meritrans DTXPlus; Merit Medical Systems, Singapore) was attached to the catheter and placed at the level of the point of the shoulder, approximating the location of the right atrium (
      • Vernemmen I.
      • Vera L.
      • Van Steenkiste G.
      • et al.
      Right atrial-related structures in horses of interest during electrophysiological studies.
      ). Heparinized saline (1 U mL–1) was used to maintain patency of the arterial catheter. Access to the facial artery was used for collection of blood for measurement of arterial blood gases, monitoring systolic (SAP), MAP and diastolic (DAP) arterial pressures and heart rate (HR) (T42; Tafonius; Hallowell Engineering & Manufacturing Corp.). All variables, except for blood gas analysis, were obtained at 5 minute intervals after positioning the horse on the table and connection to the anesthesia machine. No surgical procedures were performed on these horses. Data used for analysis were recorded from the time the treatment infusion started and were continued for the duration of anesthesia. Arterial pH, whole blood glucose and lactate concentrations, partial pressure of carbon dioxide (PaCO2) and PaO2 were measured prior to starting the treatment, on two subsequent occasions 20 minutes apart and immediately after cessation of the treatment. Arterial blood (1 mL) was collected into a 1 mL syringe (BD Medical) and immediately analyzed using a portable blood gas analyzer (i-STAT 1 Analyzer 300V; Abbott Point of Care Inc., CA, USA), a glucometer (Accu-Chek; Roche, India) and a lactate meter (Lactate Plus; Nova Biomedical, UK).

      Recovery from anesthesia

      At the end of anesthesia, controlled ventilation was discontinued and the horse was lifted using a hoist and shackle system, moved into the recovery stall and placed in right lateral recumbency on two recovery mats (each 1.2 m2 and 25 cm thick) placed side by side. Xylazine (0.2 mg kg–1) was administered IV when spontaneous breathing resumed. No supplemental oxygen was provided in the recovery stall. A camera was placed at the window of the recovery stall in a position where the horse was in full view of the camera. Video recording began after xylazine administration and the recovery room doors were closed. The same investigator (KAM) extubated all horses upon resumption of spontaneous swallowing, and all personnel left the room. Light levels in the room were not reduced and recoveries were unassisted.
      Recovery times recorded were time from positioning on the recovery mat to removal of the endotracheal tube, the time to attain sternal recumbency and number of attempts, the time to standing and number of attempts to stand by the same investigator blinded to assigned treatment (MS). After recording the recoveries, the video clips were coded and all sent to two board-certified anesthesiologists (JAEH and SAR) with more than 40 years of combined experience in equine anesthesia, and who were not involved in anesthesia or data collection. The scorers were provided with a video clip that began when xylazine was administered and ended when the horse was standing and steady on its feet. Assessment of the recoveries was performed using an equine recovery scoring system (Appendix A;
      • Donaldson L.L.
      • Dunlop G.S.
      • Holland M.S.
      • Burton B.A.
      The recovery of horses from inhalant anesthesia: a comparison of halothane and isoflurane.
      ). Recovery score <22 was considered good and a score >45 a poor recovery. All horses were returned to the MSU herd at the end of the study.

      Statistical analysis

      The sample size calculation was based on detecting a 100% increase in recovery score, representing a 14 point difference between the means of each treatment with a SD of 5 points for each treatment with an effect size of 2.8; with an alpha of p = 0.05 and a power of 80%, five horses were required (G∗Power 3.1.9.2; Heinrich-Heine-Universität Düsseldorf, Germany). An additional horse was added to increase the sample size and in the event that a horse had to be excluded from the study. Descriptive statistics were performed. Normality of the data was assessed with a Shapiro–Wilks test and a normal probability plot. Anesthesia variables, including blood gas results, were analyzed between treatments and within a treatment by means of an analysis of variance (anova) with the fixed factors of treatment, time and the random factor of horse. Data are presented as mean ± SD. Recovery data such as attempts to sternal, attempts to stand and other recovery data were compared between treatments using an anova with the fixed factor of treatment and the random factor of horse. Comparison of recovery scores of the two investigators was conducted by means of a Pearson correlation coefficient. Statistical significance was set at p ≤ 0.05.

      Results

      All six horses completed the study. Data were normally distributed with the exception of the anesthesia recovery scores, which were log transformed to obtain normality.

      Anesthesia duration and recovery

      The total anesthesia times from induction until horses were placed on the recovery mat were 82.5 ± 6.9 minutes (treatment EP) and 84.2 ± 5.8 minutes (treatment LRS) (p = 0.23). Time to extubation was 12.2 ± 2.8 minutes (treatment EP) and 16.2 ± 7.3 minutes (treatment LRS) (p = 0.22). Time to attain sternal recumbency was 24.5 ± 5.3 minutes (treatment EP) and 29.8 ± 16.2 minutes (treatment LRS) (p = 0.6). The number of attempts to attain sternal recumbency was 1.3 ± 0.5 and 1.7 ± 1.2 in treatments EP and LRS, respectively (p = 0.51). The times to standing were 39.5 ± 11.6 (treatment EP) and 39.3 ± 12.8 minutes (treatment LRS) (p = 0.92). The number of attempts to stand were 1.5 ± 0.8 and 1.8 ± 1.2 in treatments EP and LRS, respectively (p = 0.52).
      No statistically significant effect was seen between treatments EP and LRS in the quality of the recoveries based on recovery scores assigned by the two observers (p = 0.58). There was significant moderate agreement between the two observers (r2 = 0.52, p < 0.008; Table 1).
      Table 1Log mean ± standard deviation for recovery scores (<1.34, good recovery; >1.65, poor recovery) assigned by two evaluators. Horses (n = 6) were anesthetized twice with isoflurane and administered intravenous infusions of 1 L lactated Ringer’s solution (LRS) containing ethyl pyruvate (150 mg kg–1; treatment EP) or 1 L LRS (treatment LRS) over 60 minutes
      EvaluatorTreatments
      EPLRS
      11.57 ± 1.341.50 ± 1.16
      21.25 ± 0.101.21 ± 0.08

      Cardiopulmonary variables

      A dobutamine infusion was started in all horses before or about the same time that the treatments began and were administered for 15–30 minutes, until the MAP increased to 70 mmHg. Salbutamol was administered to three horses within 20 minutes of starting the treatments; to the same horse twice (once during treatment LRS and once during treatment EP), and the other two horses were in treatment EP. Actuations were delivered during the inspiratory phase of the breathing cycle.
      There were no significant differences for HR between treatments (p = 0.11). HR significantly increased from baseline at 15–55 minutes during treatment EP (p < 0.05), but HR was unchanged in treatment LRS (p > 0.05) (Table 2). There were no significant differences between treatments for SAP (p = 0.34), MAP (p = 0.75) and DAP (p = 0.52) (Table 2). SAP was increased at 10–60 minutes in both treatments (all p < 0.04). MAP increased at 20–60 minutes in treatment EP (all p < 0.02) and at 10–60 minutes in treatment LRS (all p < 0.05). DAP increased at 25–60 minutes in treatment EP (all p < 0.01) and at 15–60 minutes in treatment LRS (all p < 0.04).
      Table 2Mean ± standard deviation values for heart rate (HR), systolic arterial pressure (SAP), mean arterial pressure (MAP) and diastolic arterial pressure (DAP) in six healthy isoflurane-anesthetized horses administered infusions of ethyl pyruvate in lactated Ringer’s solution (LRS) (treatment EP) or LRS (treatment LRS) over 60 minutes during two anesthetic episodes
      VariableTreatmentTime (minutes)
      051015202530354045505560
      HR (beats minute–1)EP37 ± 341 ± 543 ± 844 ± 8∗44 ± 11∗48 ± 10∗47 ± 11∗47 ± 7∗46 ± 6∗45 ± 7∗44 ± 7∗44 ± 8∗42 ± 6
      LRS37 ± 438 ± 436 ± 337 ± 438 ± 538 ± 543 ± 842 ± 743 ± 742 ± 841 ± 739 ± 741 ± 3
      SAP (mmHg)EP81 ± 2878 ± 2095 ± 5∗97 ± 8896 ± 9∗99 ± 8∗96 ± 6∗103 ± 5∗103 ± 17∗102 ± 7∗104 ± 17∗102 ± 5∗104 ± 16∗
      LRS77 ± 1384 ± 1190 ± 13∗97 ± 14∗109 ± 16∗102 ± 11∗98 ± 12∗95 ± 9∗100 ± 10∗98 ± 10∗96 ± 8∗95 ± 6∗95 ± 9∗
      MAP (mmHg)EP58 ± 2651 ± 1670 ± 470 ± 974 ± 9∗78 ± 5∗78 ± 7∗86 ± 5∗86 ± 13∗87 ± 8∗87 ± 12∗88 ± 7∗86 ± 11∗
      LRS55 ± 1462 ± 567 ± 10∗72 ± 13∗81 ± 16∗79 ± 10∗77 ± 9∗77 ± 7∗83 ± 9∗81 ± 8∗80 ± 5∗78 ± 5∗80 ± 9∗
      DAP (mmHg)EP51 ± 1750 ± 1258 ± 461 ± 660 ± 865 ± 6∗67 ± 8∗75 ± 6∗73 ± 12∗73 ± 6∗75 ± 11∗77 ± 6∗73 ± 9∗
      LRS49 ± 751 ± 454 ± 960 ± 11∗69 ± 15∗66 ± 9∗66 ± 10∗67 ± 6∗72 ± 9∗71 ± 8∗70 ± 4∗68 ± 5∗69 ± 7∗
      ∗Significantly different from time 0 (before infusion) in the same treatment (p < 0.05).
      Whole blood glucose concentrations were not different between treatments (p = 0.25; Table 3). Blood glucose was increased from baseline at 20, 40 and 60 minutes in treatment EP (p = 0.04, p < 0.0001 and p < 0.0001, respectively), and at 40 and 60 minutes in treatment LRS (p = 0.04 and p = 0.02, respectively). There were no significant differences in PaCO2 between treatments (p = 0.62) and over time in both treatments (p > 0.05), and in PaO2 between treatments (p = 0.15) and over time in both treatments (p > 0.05). Blood lactate concentrations were different between treatments at 20, 40 and 60 minutes (p = 0.02, p < 0.0001 and p < 0.0001, respectively). Lactate concentrations progressively increased over time in treatment EP (all p < 0.0001; Table 3). Lactate concentrations were unchanged in treatment LRS (p > 0.20). Arterial pH was significantly lower at 40 and 60 minutes in treatment EP than in treatment LRS (p = 0.0001 and p = 0.01, respectively; Table 3). pH was significantly decreased from baseline in treatment EP at 20, 40 and 60 minutes (all p < 0.0001). pH was unchanged in treatment LRS (p > 0.05).
      Table 3Mean ± standard deviation values for pH, whole blood lactate and glucose concentrations, partial pressures of arterial carbon dioxide (PaCO2) and oxygen (PaO2) in six isoflurane-anesthetized horses administered infusions of ethyl pyruvate in lactated Ringer’s solution (LRS) (treatment EP) or LRS (treatment LRS) over 60 minutes during two anesthetic episodes
      VariableTreatmentTime (minutes)
      0204060
      pHEP7.39 ± 0.037.30 ± 0.04∗7.32 ± 0.02∗7.33 ± 0.03∗
      LRS7.37 ± 0.027.37 ± 0.047.38 ± 0.027.39 ± 0.02
      Lactate (mmol L–1)EP1.1 ± 0.22.5 ± 0.7∗3.2 ± 0.5∗3.8 ± 0.8∗
      LRS1.0 ± 0.51.2 ± 0.51.1 ± 0.31.3 ± 0.4
      Glucose (mg dL–1)EP119 ± 10107 ± 13∗101 ± 13∗95 ± 13∗
      LRS127 ± 17113 ± 8113 ± 20∗107 ± 19∗
      PaCO2 (mmHg; kPa)EP51.2 ± 2.657.1 ± 3.954.0 ± 3.451.4 ± 5.0
      6.8 ± 0.37.6 ± 0.57.2 ± 0.56.9 ± 0.7
      LRS54.3 ± 4.052.8 ± 5.853.2 ± 3.158.4 ± 17.3
      7.2 ± 0.57.0 ± 0.87.1 ± 0.47.8 ± 2.3
      PaO2 (mmHg; kPa)EP140 ± 77140 ± 94140 ± 52140 ± 34
      19 ± 1019 ± 1319 ± 719 ± 5
      LRS149 ± 98149 ± 146149 ± 144149 ± 119
      20 ± 1320 ± 1920 ± 1920 ± 16
      ∗Significantly different from time 0 (before infusion) in the same treatment (p < 0.05).
      Significantly different from treatment LRS at the same time point (p < 0.05).

      Discussion

      Infusion of EP for 60 minutes during isoflurane anesthesia in six healthy horses did not adversely affect measured cardiopulmonary variables or the quality of recovery from anesthesia, when compared with a control treatment of LRS.
      Studies have shown that increased anesthesia time can have a negative effect on the quality of recovery (
      • Young S.S.
      • Taylor P.M.
      Factors influencing the outcome of equine anaesthesia: a review of 1314 cases.
      ) and that horses that have had multiple anesthetic recoveries tend to recover better in subsequent events (
      • Platt J.P.
      • Simon B.T.
      • Coleman M.
      • et al.
      The effects of multiple anaesthetic episodes on equine recovery quality.
      ). Horses in the present study had prior anesthetic experiences in other research projects but none were within the past 2 years. Regardless of prior anesthetic experiences, there were no significant differences between treatments in the quality of recoveries. The
      • Donaldson L.L.
      • Dunlop G.S.
      • Holland M.S.
      • Burton B.A.
      The recovery of horses from inhalant anesthesia: a comparison of halothane and isoflurane.
      anesthesia recovery scoring system uses 10 categories to assess the quality of recovery from anesthesia in horses. This scoring system is repeatable with less than 4% interobserver variability and was found to have strong scoring correlations between 12 experienced equine anesthetists and 117 final-year veterinary students (
      • Vettorato E.
      • Chase-Topping M.E.
      • Clutton R.E.
      A comparison of four systems for scoring recovery quality after general anaesthesia in horses.
      ).
      The anesthetists who scored the video clips of recovery in the current study read the paper by
      • Donaldson L.L.
      • Dunlop G.S.
      • Holland M.S.
      • Burton B.A.
      The recovery of horses from inhalant anesthesia: a comparison of halothane and isoflurane.
      describing the recovery scoring system, and no further training was performed.
      • Clark-Price S.C.
      • Posner L.P.
      • Gleed R.D.
      Recovery of horses from general anesthesia in a darkened or illuminated recovery stall.
      used the same scoring system but added an 11th category that summated the recovery into an overall recovery score. In the present study, the assigned recovery scores were compared between evaluators and between treatments, with no differences being noted. A possible reason for these findings was that all horses were administered the same dosage of xylazine IV in recovery, once spontaneously breathing. This practice has been shown to help improve recovery as horses may try to stand too quickly once the effect of the inhalant diminishes (
      • Hubbell J.A.E.
      Horses.
      ). It is possible that EP could affect the rate of elimination of the inhalant as it has been shown in rats to improve CO, thereby enhancing the delivery of inhalant to the lungs (
      • Taylor M.D.
      • Grand T.J.
      • Cohen J.E.
      • et al.
      Ethyl pyruvate enhances ATP levels, reduces oxidative stress and preserves cardiac function in a rat model of off-pump coronary bypass.
      ). No studies have reported the effect of EP on CO in horses. Xylazine administered to the horses during recovery in the present study could have masked any negative effect of EP. However, because it is standard practice and there are data to support its use, xylazine was administered to all of the horses when they were placed in the recovery stall (
      • Hubbell J.A.E.
      Horses.
      ). Another possible reason for a lack of difference in the recovery scores is that the horses in this study had previously undergone anesthetic recoveries for other studies and may have recovered better as a result of their experience (
      • Platt J.P.
      • Simon B.T.
      • Coleman M.
      • et al.
      The effects of multiple anaesthetic episodes on equine recovery quality.
      ). This possible limitation was minimized by anesthetizing the horses twice with treatments administered in a random order.
      HR did not change significantly between treatments but increased over time in treatment EP. The increased HR was not clinically significant as it was within the normal range for horses (
      • McConachie E.L.
      • Giguère S.
      • Rapoport G.
      • Barton M.H.
      Heart rate variability in horses with acute gastrointestinal disease requiring exploratory laparotomy.
      ). All horses were ventilated using controlled ventilation after induction of anesthesia and as such, PaCO2 did not change significantly over time or between treatments. All horses were hypotensive from the start of isoflurane anesthesia and were administered a dobutamine infusion of 1–5 μg kg–1 minute–1 for 15–30 minutes to maintain a MAP >70 mmHg (
      • Donaldson L.L.
      Retrospective assessment of dobutamine therapy for hypotension in anesthetized horses.
      ;
      • Driessen B.
      • Nann L.
      • Benton R.
      • Boston R.
      Differences in need for hemodynamic support in horses anesthetized with sevoflurane as compared to isoflurane.
      ;
      • Loughran C.M.
      • Raisis A.L.
      • Hosgood G.
      • et al.
      The effect of dobutamine and bolus crystalloid fluids on the cardiovascular function of isoflurane-anaesthetized horses.
      ). The progressive increase in arterial pressure, without a difference between treatments, could be attributed to the use of dobutamine.
      Salbutamol was used during four of the 12 anesthetic episodes in the current study. In three horses, PaO2 <90 mmHg (12 kPa) was measured before the treatments began and were not associated with EP treatment. In another horse, PaO2 <70 mmHg (12 kPa) was measured at 20 minutes after the start of the EP treatment.
      Blood glucose concentrations decreased over time during both treatments but the changes were not clinically significant. Blood lactate concentrations increased from baseline at all time points measured first at 20 minutes in treatment EP. The decrease in arterial pH in treatment EP can probably be attributed to the low pH of the EP solution (pH < 7.0) and the increased lactate concentrations, because these changes did not occur in the LRS treatment. Pyruvate is a lactate precursor and lactate is a metabolite of EP, which could result in acidosis (
      • Yang R.
      • Zhu S.
      • Tonnessen T.I.
      Ethyl pyruvate is a novel anti-inflammatory agent to treat multiple inflammatory organ injuries.
      ). These findings agree with previously reported studies showing that EP can be degraded and converted to pyruvate, and then to lactate (
      • Jacobs C.C.
      • Holcombe S.J.
      • Cook V.L.
      • et al.
      Ethyl pyruvate diminishes the inflammatory response to lipopolysaccharide infusion in horses.
      ;
      • Yang R.
      • Zhu S.
      • Tonnessen T.I.
      Ethyl pyruvate is a novel anti-inflammatory agent to treat multiple inflammatory organ injuries.
      ). Increasing concentrations of lactate can lead to a decrease in pH which fits with the findings in this study (
      • Koprivica I.
      • Gajić D.
      • Pejnović N.
      • et al.
      Ethyl pyruvate promotes proliferation of regulatory T cells by increasing glycolysis.
      ). The increases in lactate concentrations during treatment EP in the current study were not clinically significant.
      There were several limitations in this study. Healthy hemodynamically normal horses of the same breed and of the same ASA classification were studied. Treatment EP was only 60 minutes, which may not have been long enough for clinically significant changes to develop. Administration of EP over a longer time or at a higher dosage may induce different effects. Horses with metabolic abnormalities may develop further changes during EP administration when compared with healthy horses. CO was not measured, which would have provided more information about the effect of EP on cardiovascular function in anesthetized horses. A further limitation was the use of clinical signs to determine anesthetic depth rather than measurement of end-tidal isoflurane concentration.

      Conclusions

      Administration of EP (150 mg kg–1 over 60 minutes) to healthy isoflurane-anesthetized horses resulted in no negative effects on the cardiopulmonary variables studied or on the quality of the recovery from anesthesia. Further studies of EP are necessary to ensure that adverse effects are not induced in hemodynamically unstable horses.

      Acknowledgements

      The authors thank Dr Joe G Hauptman, College of Veterinary Medicine, Michigan State University, for providing the statistical analysis and assisted with manuscript preparation. This study was funded by the Freeman Fund for equine research, College of Veterinary Medicine Endowed Research Funds, Michigan State University .

      Authors’ contributions

      KAM, MS and SJH: study design, data collection, preparation of manuscript. JMM: assisted with statistical analysis, manuscript preparation. SAR and JAEH: scored anesthesia recoveries, preparation of manuscript. All authors read and approved the final version of the manuscript.

      Conflict of interest statement

      The authors declare no conflicts of interest.

      Appendix A. Scoring recovery from anesthesia

      Tabled 1
      BehaviorScoreDescription
      Overall attitude (1–10)1Calm
      3Calm/determined
      5Anxious
      7Confused/dizzy
      8Angry
      10Frantic
      Activity in recumbency (1–5)1Quiet, occasional stretch, head lift
      3Tense, waiting to explode
      5Flailing
      Move to sternal (1–10)1Smooth, methodical
      5Fighting mat but controlled
      10Crashing, flopping over
      Number of attempts to sternal (score = #)
      Sternal phase (1–10)1An organized pause
      3Non-existent
      6Prolonged
      7Multiple
      10Continues to struggle
      Move to stand (1–10)1Methodical
      3An organized scramble
      6Used walls for support
      10Ricocheting off walls
      Strength (1–10)1Near full
      3Mildly rubbery
      6Dog sitting before standing
      10Repeated attempts because of weakness
      Number of attempts to stand (score = #)
      Balance and Coordination (1–10)1Solid
      3Moderate dancing
      5Reflex saves
      8Careening
      10Falls back down
      Knuckling (1–5)1None
      2Hind limbs only – mild
      3Hind limbs only – marked
      4All four – moderate
      5Excessive, prolonged
      Reprinted from
      • Donaldson L.L.
      • Dunlop G.S.
      • Holland M.S.
      • Burton B.A.
      The recovery of horses from inhalant anesthesia: a comparison of halothane and isoflurane.
      with permission. ©John Wiley & Sons Inc.

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